This invention relates to novel polypeptides and their derivatives which act as inhibitors or regulators of complement activation and are of use in the therapy of diseases involving complement activation such as various inflammatory and immune disorders.
Constituting about 10% of the globulins in normal serum, the complement system is composed of many different proteins that are important in the immune system""s response to foreign antigens. The complement system becomes activated when its primary components are cleaved and the products alone or with other proteins, activate additional complement proteins resulting in a proteolytic cascade. Activation of the complement system leads to a variety of responses including increased vascular permeability, chemotaxis of phagocytic cells, activation of inflammatory cells, opsonization of foreign particles, direct killing of cells and tissue damage. Activation of the complement system may be triggered by antigen-antibody complexes (the classical pathway) or, for example, by lipopolysaccharides present in cell walls of pathogenic bacteria (the alternative pathway).
Complement activation (CA) is known to occur in a wide variety of acute inflammatory processes particularly those associated with ischaemia and reperfusion injury (Rossen et al., 1985 Circ. Res., 57, 119,; Morgan B. P., 1990 The biological effects of complement activation. In xe2x80x98Complement, Clinical Aspects and Relevance to Diseasexe2x80x99. Academic Press. London.)
It is also generally accepted that at least some of the components of the classical complement cascade can be detected by immunohistochemical methods in close association with senile plaques in the brains of sufferers from Alzheimer""s disease (Eikelenboom et al., 1994, Neuroscience, 59, 561-568) and that complement activation plays a role in the inflammatory component of this condition.
Complement receptor type 1 (CR1) has been shown to be present on the membranes of erythrocytes, monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes. CR1 binds to the complement components C3b and C4b and has also been referred to as the C3b/C4b receptor. The structural organisation and primary sequence of one allotype of CR1 is known (Klickstein et al., 1987, J. Exp. Med. 165:1095-1112, Klickstein et al., 1988, J. Exp. Med. 168:1699-1717; Hourcade et al., 1988, J. Exp. Med. 168:1255-1270, WO 89/09220, WO 91/05047). It is composed of 30 short consensus repeats (SCRs) that each contain around 60-70 amino acids. In each SCR, around 29 of the average 65 amino acids are conserved. Each SCR has been proposed to form a three dimensional triple loop structure through disulphide linkages with the third and first and the fourth and second half-cystines in disulphide bonds. CR1 is further arranged as 4 long homologous repeats (LHRs) of 7 SCRs each. Following a leader sequence, the CR1 molecule consists of the N-terminal LHR-A, the next two repeats, LHR-B and LHR-C, and the most C-terminal LHR-D followed by 2 additional SCRs, a 25 residue putative transmembrane region and a 43 residue cytoplasmic tail.
Based on the mature CR1 molecule having a predicted N-terminal glutamine residue, hereinafter designated as residue 1, the first four SCR domains of LHR-A are defined herein as consisting of residues 2-58, 63-120, 125-191 and 197-252, respectively, of mature CR1.
Hourcade et al., 1988, J. Exp. Med. 168:1255-1270 observed an alternative polyadenylation site in the human CR1 transcriptional unit that was predicted to produce a secreted form of CR1. The mRNA encoded by this truncated sequence comprises the first 8.5 SCRs of CR1, and encodes a protein of about 80 kDa which was proposed to include the C4b binding domain. When a cDNA corresponding to this truncated sequence was transfected into COS cells and expressed, it demonstrated the expected C4b binding activity but did not bind to C3b (Krych et al., 1989, FASEB J. 3:A368; Krych et al. Proc. Nat. Acad. Sci. 1991, 88, 4353-7). Krych et al., also observed a mRNA similar to the predicted one in several human cell lines and postulated that such a truncated soluble form of CR1 with C4b binding activity may be synthesised in humans.
In addition, Makrides et al. (1992, J. Biol. Chem. 267 (34) 24754-61) have expressed SCR 1+2 and 1+2+3+4 of LHR-A as membrane-attached proteins in CHO cells.
Several soluble fragments of CR1 have also been generated via recombinant DNA procedures by eliminating the transmembrane region from the DNAs being expressed (WO 89/09220, WO 91/05047). The soluble CR1 fragments were functionally active, bound C3b and/or C4b and demonstrated Factor I cofactor activity depending upon the regions they contained. Such constructs inhibited in vitro complement-related functions such as neutrophil oxidative burst, complement mediated hemolysis, and C3a and C5a production. A particular soluble construct, sCR1/pBSCR1c, also demonstrated in vivo activity in a reversed passive Arthus reaction (WO 89/09220, WO 91/05047; Yeh et al., 1991, J. Immunol. 146:250), suppressed post-ischemic myocardial inflammation and necrosis (WO 89/09220, WO 91/05047; Weisman et al., Science, 1990, 249:146-1511, Dupe, R. et al. Thrombosis and Haemostasis (1991) 65(6) 695.) and extended survival rates following transplantation (Pruitt and Bollinger, 1991, J. Surg. Res 50:350; Pruitt et al., 1991 Transplantation 52; 868). Furthermore, co-formulation of sCR1/pBSCR1c with p-anisoylated human plasminogen-streptokinase-activator complex (APSAC) resulted in similar anti-haemolytic activity as sCR1 alone, indicating that the combination of the complement inhibitor sCR1 with a thrombolytic agent was feasible (WO 91/05047).
In a model of antibody-mediated demyelinating experimental allergic encephalomyclitis (ADEAE), systemic inhibition of CA using sCR1 over 6 days, produced improvements in clinical score and blocked CNS inflammation, demyelination and deposition of complement components (Piddlesden et al., 1994, J. Immunol. 152, 5477). ADEAE can be regarded as a model of acute relapse in multiple sclerosis (MS) and these striking results suggested possible applications for sCR1 in MS therapy despite the high molecular weight (245 kilodaltons) of this agent.
In a rat model of traumatic brain injury, complement inhibitor sCR1 (also known as TP10 or BRL55730) was shown to reduce myeloperoxidase activity (an indicator of neutrophil accumulation) following traumatic injury (Kaczorowska et al, 1995, J. Cerebral Blood Flow and Metabolism, 15, 860-864). This is suggested as demonstrating that complement activation is involved in the local inflammatory response.
Soluble polypeptides corresponding to part of CR1 having functional complement inhibitory, including anti-haemolytic activity, have been described in WO94/00571 comprising, in sequence, one to four short consensus repeats (SCR) selected from SCR 1, 2, 3 and 4 of long homologous repeat A (LHR-A) as the only structurally and functionally intact SCR domains of CR1 and including at least SCR3.
Pseudogenes are usually defined as DNA sequences which possess a high degree of homology to genes with identified function but which are not expressed. The origins of the lack of transcription and translation vary but are commonly the presence of accumulated mutations which inactivate inscriptional initiation sites, disrupt RNA splicing or introduce frame-shift mutations and premature termination codons. Pseudogenes are sometimes regarded as genetic relics which have been isolated within the genome through a primary loss of expressability and which have subsequently mutated randomly in situ to highly aberrant forms. There is a frequent presumption that pseudogene sequences, if expressable at all, will not be functionally active because of an accumulation of deleterious in-frame mutations. However, studies of immune system genetics suggest that pseudogenes may act as a source of diversity in somatic mutation processes and that non-expressed sequences may recombine with normally expressed genes to create functional variants with a conserved framework. This phenomenon has been documented in immunoglobulin VL and VH genes and elsewhere (W. T. McCormack et al., Genes Dev. 4, 548-58, 1990).
The creation of pseudogenes through reverse transcription followed by DNA integration is also known. In such cases, the integrated sequences (which can in principle originate from organisms other than the host) lack introns and may be sited in chromosomal locations distant from the expressed gene to which they are homologous because integration into the genome can occur at random sites. Such genes are known as processed pseudogenes. The presence of pseudogenes in chromosomal clusters with homologous expressed genes argues against them being processed pseudogenes because of the improbability of a random integration process giving rise to close physical clustering in a large genome.
The existence of a gene homologous to that for complement receptor type 1 (CR1) was first reported by Hourcade et al. (J.Biol.Chem, 275, 974-80, 1990), who found it associated with a gene cluster on chromosome 1q 32 termed the Regulators of Complement Activation (RCA) cluster. The latter comprises the CR1 gene itself and those encoding decay accelerating factor (DAF), membrane cofactor protein (MCP), Factor H, complement receptor type 2 (CR2) and C4 binding protein. This xe2x80x98CR1-likexe2x80x99 gene was predicted to encode a protein containing seven SCR regions corresponding (by closest homology) to SCRs 1-6 and 9 of LHR-A (1-6) or LHR-B (9) of CR1 itself. The overall homology with the above regions of CR1 at the predicted amino acid level is 91% and the sequence divergence is greatest in the first three SCRs.
The CR1-like gene also encodes a signal peptide but not a transmembrane or cytoplasmic region and contains an intron-exon structure similar to the CR1 gene. There is currently no evidence that the CR1-like gene is expressed and neither mRNA transcripts nor a soluble protein have been isolated. The origin of the CR1-like sequence may lie in a gene duplication event in an ancestral CR1 gene which was followed by divergence and transcriptional inactivation of the CR1-like gene. It appears probable that the CR1-like gene is currently a pseudogene although not of the processed type.
It has now been found that replacement of codons in DNA encoding the first three SCRs of LHR-A of CR1 with others encoding the predicted aminoacids in the CR1-like sequence can give rise to chimeric genes which can be expressed to give active complement inhibitors with functional complement inhibitory, including anti-haemolytic, activity.
According to the present invention there is provided a soluble polypeptide comprising, in sequence, one to four short consensus repeats (SCR) selected from SCR 1, 2, 3 and 4 of long homologous repeat A (LHR-A) as the only structurally and functionally intact SCR domains of CR1 and including at least SCR3, in which one or more of the native amino acids are substituted with the following:
Val 4, Asp 19, Ser 53, Lys 57, Ala 74, Asp 79, Arg 84, Pro 91, Asn 109, Lys 116, Val 119, Ala 132, Thr 137, Ile 139, Ser 140, Tyr 143, His 153, Leu 156, Arg 159, Lys 161, Lys 177, Gly 230, Ser 235, His 236.
(Numbering is from glutamine as residue 1 of mature CR1. The amino-acid indicated is that which replaces the CR1 residue at the position specified.).
In preferred aspects, the polypeptide comprises, in sequence, SCR 1, 2, 3 and 4 of LHR-A or SCR 1, 2 and 3 of LHR-A as the only structurally and functionally intact SCR domains of CR1 with the modification(s) described above.
It is to be understood that additional variations in the amino acid sequence of the polypeptide of the invention by way of addition, deletion or conservative substitution of residues, including allelic variations, in which the biological activity of the polypeptide is retained, are encompassed by the invention. Conservative substitution is understood to mean the retention of the charge and/or size characteristics of the amino acid side chain, for example arginine replaced by lysine or glutamine.
In one aspect, the polypeptide of the invention may be represented symbolically as follows:
NH2-V1-SCR1-W1-SCR2-X1-SCR3-Y1-OHxe2x80x83xe2x80x83(I)
in which SCR1 represents residues 2-58 of mature CR1, SCR2 represents residues 63-120 of mature CR1, SCR3 represents residues 125-191 of mature CR1, and containing at least one of the substitutions as aforesaid and V1, W1, and Y1 represent bonds or short linking sequences of amino acids, preferably 1 to 5 residues in length and which are preferably derived from native interdomain sequences in CR1.
The native interdomain sequences in CR1 may also be substituted with the corresponding predicted aminoacids in the CR1-like sequence, namely Lys59 and/or Ile124. (Numbering is from glutamine as residue 1 of mature CR1. The amino-acid indicated is that which replaces the CR1 residue at the position specified.)
In a preferred embodiment, the SCR3 domain of formula (I) is substituted with all ten residues found in the corresponding pseudogene sequence, namely (in single letter code):
A132, T137, I139, S140, Y143, H153, L156, R159, K161, K177xe2x80x83xe2x80x83(Sequence Group 1)
and the remaining domains have the sequence of mature CRI.
In a further preferred embodiment of formula (I), W1, X1 and Y1 represent residues 59-62, 121-124 and 192-196, respectively, of mature CR1, optionally substituted as aforesaid and V1 represents residue 1 of mature CR1 optionally linked via its N-terminus to methionine.
In another aspect the polypeptide of the invention may be represented symbolically as follows:
NH2-V2-SCR1-W2-SCR2-X2-SCR3-Y2-SCR4-Z2OHxe2x80x83xe2x80x83(II)
in which SCR1, SCR2 and SCR3 are as hereinbefore defined, SCR4 represents residues 197-252 of mature CR1 and containing at least one of the substitutions as aforesaid, and V2, W2, X2, Y2 and Z2 represent bonds or short linking sequences of amino acids, preferably 1 to 5 residues in length and which are preferably derived from native interdomain sequences in CR1, optionally substituted as aforesaid.
In a preferred embodiment of formula (II)), the SCR3 region is substituted with the aforementioned Sequence Group 1 residues and the remaining domainshave the sequence of mature CR1.
In further preferred embodiments of formula (II), W2, X2, Y2 and Z2 represent residues 59-62, 121-124, 192-196, and residues 253 respectively, of mature CR1, optionally substituted as aforesaid, and V2 represents residue 1 of mature CR1 optionally linked via its N-terminus to methionine.
In one particular embodiment of formula (II) arginine 235 is replaced by histidine.
In the preferred embodiment of formula (II), residue 235 is arginine.
In one further aspect, the polypeptide of the invention may be represented symbolically as follows:
NH2-X3-SCR3-Y3-OHxe2x80x83xe2x80x83(III)
in which SCR3 is as hereinbefore defined, containing at least one of the substitutions as aforesaid, and in a preferred embodiment, all those of Sequence Group 1, and X3 and Y3 represent bonds or short linking sequences of amino acids, preferably 1 to 5 residues in length and which are preferably derived from native interdomain sequences in CR1, optionally substituted as aforesaid.
In a further preferred embodiment of formula (III) X3 represents amino acids 122-124 of mature CR1, optionally substituted as aforesaid, optionally linked to methionine at its N-terminus and Y4 represents amino acids 192-196 of mature CR1.
In another further aspect, the polypeptide of the invention may be represented symbolically as follows:
NH2-X4-SCR3-Y4-SCR4-Z4-OHxe2x80x83xe2x80x83(IV)
in which SCR3 and SCR4 are as hereinbefore defined containing at least one of the substitutions as aforesaid and X4, Y4 and Z4 represent bonds or short linking sequences of amino acids, preferably 1 to 5 residues in length and which are preferably derived from native interdomain sequences in CR1, optionally substituted as aforesaid.
In a preferred embodiment of formula (IV), the SCR3 region is substituted with the residues of Sequence Group 1 and the remaining domainshave the sequence of mature CR1.
In a further preferred embodiment of formula (IV) X4 represents amino acids 122-124 of mature CR1, optionally substituted as aforesaid, optionally linked to methionine at its N-terminus and Y4 and Z4 represent amino acids 192-196 and 253 respectively of mature CR1.
The soluble polypeptides of the invention lack the membrane binding capability of the full length CR1 proteins which properties may be advantageous to the therapeutic activity.
The main classes of interaction of proteins with membranes can be summarised as follows:
1. Direct and specific interactions with phospholipid head groups or with other hydrophilic regions of complex lipids or indirectly with proteins already inserted in the membrane. The latter may include all the types of intrinsic membrane protein noted below and such interactions are usually with extracellular domains or sequence loops of the membrane proteins;
2. Through anchoring by a single hydrophobic transmembrane helical region near the terminus of the protein. These regions commonly present a hydrophobic face around the entire circumference of the helix cylinder and transfer of this structure to the hydrophilic environment of bulk water is energetically unfavourable.
3. Further anchoring is often provided by a short sequence of generally cationic aminoacids at the cytoplasmic side of the membrane, C-terminal to the transmembrane helix. The membrane-binding properties or CR1 are provided by features 2 and 3.
4. Through the use of multiple (normally 2-12 and commonly 4, 7 and 10) transmembrane regions which are usually predicted to be helical or near-helical. Although these regions are normally hydrophobic overall, they frequently show some amphipathic behaviourxe2x80x94an outer hydrophobic face and an inner more hydrophilic one being identifiable within a helix bundle located in the lipid bilayer;
5. Through postranslationally linked phosphatidyl inositol moieties (GPI-anchors). These are generated by a specific biosynthetic pathway which recognises and removes a specific stretch of C-terminal aminoacids and creates a membrane-associating diacyl glycerol unit linked via a hydrophilic carbohydrate spacer to the polypeptide;
6. In a related process, single fatty acid groups such as myristoyl, palmitoyl or prenyl may be attached postranslationally to one or more sites in a protein (usually at N- or C-termini). Again, amino acids (such as the C-terminal CAAX box in Ras proteins) may be removed.
The present invention further provides a soluble derivative of the soluble polypeptide of the invention, said derivative comprising two or more heterologous membrane binding elements with low membrane affinity covalently associated with the polypeptide which elements are capable of interacting independently and with thermodynamic additivity with components of cellular membranes exposed to extracellular fluids.
By xe2x80x98heterologousxe2x80x99 is meant that the elements are not found in the native full length CR1 protein.
By xe2x80x98membrane binding element with low membrane affinityxe2x80x99 is meant that the element has significant affinity for membranes, that is a dissociation constant greater than 1 xcexcM, preferably 1 xcexcM-1 mM. The elements preferably have a size  less than 5 kDa.
The derivative should incorporate sufficient elements with low affinities for membrane components to result in a derivative with a high (preferably 0.01-10 nM dissociation constant) affinity for specific membranes. The elements combine so as to create an overall high affinity for the particular target membrane but the combination lacks such high affinity for other proteins for which single elements may be (low-affinity) ligands.
The elements should be chosen so as to retain useful solubility in pharmaceutial formulation media, preferably  greater than 100 xcexcg/ml. Preferably at least one element is hydrophilic.
The further embodiment of the invention thus promotes localisation of the polypeptide of the invention at cellular membranes and thereby provide one or more of several biologically significant effects with potential therapeutic advantages including:
Potency: an increase in effective concentration may result from the reduction in the diffusional degrees of freedom.
Pharmacokinetics and dosing frequency: Interaction of the derivatised polypeptide with long-lived cell types or serum proteins would be expected to prolong the plasma residence time of the polypeptide and produce a depot effect through deposition on cell surfaces.
Specificity: Many clinically important pathological processes are associated with specific cell types and tissues (for example the vascular endothelium and the recruitment thereto of neutrophils bearing the sialyl Lewisx antigen to ELAM-1, see below). Hence targeting the modified polypeptide to regions of membrane containing pathology-associated membrane markers may improve the therapeutic ratio of the protein targeted.
It will be appreciated that all associations of heterologous amino acid sequences with the polypeptide will need to be assessed for potential immunogenicity, particularly where the amino acid sequence is not derived from a human protein. The problem can be minimised by using sequences as close as possible to known human ones and through computation of secondary structure and antigenicity indices.
The derivative preferably comprises two to eight, more preferably two to four membrane binding elements.
Membrane binding elements are preferably selected from: fatty acid derivatives such as fatty acyl groups; basic amino acid sequences; ligands of known integral membrane proteins; sequences derived from the complementarity-determining region of monoclonal antibodies raised against epitopes of membrane proteins; membrane binding sequences identified through screening of random chemical libraries.
The selection of suitable combination of membrane binding elements will be guided by the nature of the target cell membrane or components thereof.
Suitable fatty acid derivatives include myristoyl (12 methylene units) which is insufficiently large or hydrophobic to permit high affinity binding to membranes. Studies with myristoylated peptides (eg R. M. Peitzsch and S. McLaughlin, Biochemistry, 32, 10436-10443, 1993)) have shown that they have effective dissociation constants with model lipid systems of xcx9c10xe2x88x924 M and around 10 of the 12 methylene groups are buried in the lipid bilayer. Thus, aliphatic acyl groups with between about 8 and 18 methylene units, preferably 10-14, are suitable membrane binding elements. Other examples of suitable fatty acid derivatives include long-chain (8-18, preferably 10-14 methylene) aliphatic amines and thiols, steroid and farnesyl derivatives.
Membrane binding has been found to be associated with limited (single-site) modification with fatty acyl groups when combined with a cluster of basic aminoacids in the protein sequence which may interact with acidic phospholipid head groups and provide the additional energy to target membrane binding. This combination of effects has been termed the xe2x80x98myrstoyl-electrostatic switchxe2x80x99 (S. McLaughlin and A. Aderem, TIBS, 20,272-276, 1994; J. F. Hancock et al, Cell, 63, 133-139,1990). Thus, a further example of suitable membrane binding elements are basic aminoacid sequences such as those found in proteins such as Ras and MARCKS (myristoylated alanine-rich C-kinase substrate, P. J. Blackshear, J. Biol. Chem., 268, 1501-1504, 1993) which mediate the electrostatic xe2x80x98switchxe2x80x99 through reversible phosphorylation of serine residues within the sequence and a concomitant neutralisation of the net positive charge. Such sequences include but are not restricted to consecutive sequences of Lysine and Arginine such as (Lys)n where n is between 3 and 10, preferable 4 to 7.
Suitable examples of amino acid sequences (SEQ ID NOS 60, 61, and 62 respectively) comprising basic amino acids include:
DGPKKKKKKSPSKSSGxe2x80x83xe2x80x83i)
GSSKSPSKKKKKKPGDxe2x80x83xe2x80x83ii)
SPSNETPKKKKKRFSFKKSGxe2x80x83xe2x80x83iii)
(N-terminus on left)
Sequences i) to iii) are examples of electrostatic switch sequences.
Examples of amino acid sequences include RGD-containing peptides such as GRGDSP (SEQ ID NO:63) which are ligands for the xcex1libxcex2; integrin of human platelet membranes. Further examples of such sequences include those known to be involved in interactions between membrane proteins such as receptors and the major histocompatibility complex. An example of such a membrane protein ligand is the sequence (SEQ ID NO:64) GNEQSFRVDLRTLLRYA which has been shown to bind to the major histocompatibility complex class 1 protein (MHC-1) with moderate affinity (L. Olsson et al, Proc. Natl. Acad.Sci.USA. 91, 9086-909, 1994).
An example of a ligand for an integral membrane protein is the carbohydrate ligand Sialyl Lewisx which has been identified as a ligand for the integral membrane protein ELAM-1 (M. L. Phillips et al, Science, 250, 1130-1132, 1990 and G. Walz et al, Ibid. 250, 1132-1135,1990).
Sequences derived from the complementarity-determining regions of monoclonal antibodies raised against epitopes within membrane proteins (see, for example, J. W. Smith et al, J.Biol.Chem. 270, 30486-30490, 1995) are also suitable membrane binding elements, as are binding sequences from random chemical libraries such as those generated in a phage display format and selected by biopanning operations in vitro (G. F. Smith and J. K. Scott, Methods in Enzymology, 217H, 228-257,1993) or in vivo (R. Pasqualini and E. Ruoslahti, Nature, 380, 364-366, 1996).
Optionally, conditional dissociation from the membrane may be incorporated into derivatives of the invention using mechanisms such as pH sensitivity (electrostatic switches), regulation through metal ion binding (using endogenous Ca2+, Z2+ and incorporation of ion binding sites in membrane binding elements) and protease cleavage (e.g plasminolysis of lysine-rich membrane binding sequences to release and activate prourokinase)
Preferred derivatives of this invention have the following structure:
[P]-{L-[W]}n-X
in which:
P is the soluble polypeptide,
each L is independently a flexible tinker group,
each W is independently a peptide membrane binding element,
n is an integer of 1 or more and
X is a peptide or non-peptide membrane-binding entity which may be covalently linked to any W.
Peptide membrane binding elements are preferably located sequentially either at the N or C terminus of the soluble polypeptide and are preferably 8 to 20 amino acids long. The amino acid sequences are linked to one another and to the soluble peptide by linker groups which are preferably selected from hydrophilic and/or flexible aminoacid sequences of 4 to 20 aminoacids; linear hydrophilic synthetic polymers; and chemical bridging groups.
In a further aspect, the invention provides a process for preparing a polypeptide according to the invention which process comprises expressing DNA encoding said polypeptide in a recombinant host cell and recovering the product.
In particular, the process may comprise the steps of:
i) preparing a replicable expression vector capable, in a host cell, of expressing a DNA polymer comprising a nucleotide sequence that encodes said polypeptide;
ii) transforming a host cell with said vector;
iii) culturing said transformed host cell under conditions permitting expression of said DNA polymer to produce said polypeptide; and
iv) recovering said polypeptide.
The DNA polymer comprising a nucleotide sequence that encodes the polypeptide also forms part of the invention.
The process of the invention may be performed by conventional recombinant techniques such as described in Sambrook et al., Molecular Cloning: A laboratory manual 2nd Edition. Cold Spring Harbor Laboratory Press (1989) and DNA Cloning vols I, II and III (D. M. Glover ed., IRL Press Ltd).
The invention also provides a process for preparing the DNA polymer by the condensation of appropriate mono-, di- or oligomeric nucleotide units.
The preparation may be carried out chemically, enzymatically, or by a combination of the two methods, in vitro or in vivo as appropriate. Thus, the DNA polymer may be prepared by the enzymatic ligation of appropriate DNA fragments, by conventional methods such as those described by D. M. Roberts et al., in Biochemistry 1985, 24, 5090-5098.
The DNA fragments may be obtained by digestion of DNA containing the required sequences of nucleotides with appropriate restriction enzymes, by chemical synthesis, by enzymatic polymerisation, or by a combination of these methods.
Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20xc2x0-70xc2x0 C., generally in a volume of 50 xcexcl or less with 0.1-10 xcexcg DNA.
Enzymatic polymerisation of DNA may be carried out in vitro using a DNA polymerase such as DNA polymerase 1 (Klenow fragment) in an appropriate buffer containing the nucleoside triphosphates dATP, dCTP, dGTP and dTTP as required at a temperature of 10xc2x0-37xc2x0 C., generally in a volume of 50 xcexcl or less.
Enzymatic ligation of DNA fragments may be carried out using a DNA ligase such as T4 DNA ligase in an appropriate buffer at a temperature of 4xc2x0 C. to 37xc2x0 C., generally in a volume of 50 xcexcl or less.
The chemical synthesis of the DNA polymer or fragments may be carried out by conventional phosphotriester, phosphite or phosphoramidite chemistry, using solid phase techniques such as those described in xe2x80x98Chemical and Enzymatic Synthesis of Gene Fragmentsxe2x80x94A Laboratory Manualxe2x80x99 (ed. H. G. Gassen and A. Lang), Verlag Chemie, Weinheim (1982), or in other scientific publications, for example M. J. Gait, H. W. D. Matthes M. Singh, B. S. Sproat and R. C. Titmas, Nucleic Acids Research, 1982, 10, 6243; B. S. Sproat and W. Banawarth, Tetrahedron Letters, 1983, 24, 5771; M. D. Matteucci and M. H. Caruthers, Tetrahedron Letters, 1980, 21, 719; M. D. Matteucci and M. H. Caruthers, Journal of the American Chemical Society, 1981, 103, 3185; S. P. Adams et al., Journal of the American Chemical Society, 1983, 105, 661; N. D. Sinha, J. Biernat, J. McMannus and H. Koester, Nucleic Acids Research, 1984, 12, 4539; and H. W. D. Matthes et al., EMBO Journal, 1984, 3, 801. Preferably an automated DNA synthesiser (for example, Applied Biosystems 381A Synthesiser) is employed.
The DNA polymer is preferably prepared by ligating two or more DNA molecules which together comprise a DNA sequence encoding the polypeptide.
The DNA molecules may be obtained by the digestion with suitable restriction enzymes of vectors carrying the required coding sequences.
The precise structure of the DNA molecules and the way in which they are obtained depends upon the structure of the desired product. The design of a suitable strategy for the construction of the DNA molecule coding for the polypeptide is a routine matter for the skilled worker in the art.
In particular, consideration may be given to the codon usage of the particular host cell. The codons may be optimised for high level expression in E. coli using the principles set out in Devereux et al., (1984) Nucl. Acid Res., 12, 387.
The expression of the DNA polymer encoding the polypeptide in a recombinant host cell may be carried out by means of a replicable expression vector capable, in the host cell, of expressing the DNA polymer. The expression vector is novel and also forms part of the invention.
The replicable expression vector may be prepared in accordance with the invention, by cleaving a vector compatible with the host cell to provide a linear DNA segment having an intact replicon, and combining said linear segment with one or more DNA molecules which, together with said linear segment, encode the polypeptide, under ligating conditions.
The ligation of the linear segment and more than one DNA molecule may be carried out simultaneously or sequentially as desired.
Thus, the DNA polymer may be preformed or formed during the construction of the vector, as desired. The choice of vector will be determined in part by the host cell, which may be prokaryotic, such as E. coli, or eukaryotic, such as mouse C127, mouse myeloma chinese hamster ovary, fungi e.g. filamentous fungi or unicellular xe2x80x98yeastxe2x80x99 or an insect cell such as Drosophila. The host cell may also be in a transgenic animal. Suitable vectors include plasmids, bacteriophages, cosmids and recombinant viruses derived from, for example, baculoviruses or vaccinia.
The DNA polymer may be assembled into vectors designed for isolation of stable transformed mammalian cell lines expressing the fragment e.g. bovine papillomavirus vectors in mouse C127 cells, or amplified vectors in chinese hamster ovary cells (DNA Cloning Vol. II D. M. Glover ed. IRL Press 1985; Kaufman, R. J. et al., Molecular and Cellular Biology 5, 1750-1759, 1985; Pavlakis G. N. and Hamer, D. H. Proceedings of the National Academy of Sciences (USA) 80, 397-401, 1983; Goeddel, D. V. et al., European Patent Application No. 0093619, 1983).
The preparation of the replicable expression vector may be carried out conventionally with appropriate enzymes for restriction, polymerisation and ligation of the DNA, by procedures described in, for example, Sambrook et al., cited above. Polymerisation and ligation may be performed as described above for the preparation of the DNA polymer. Digestion with restriction enzymes may be performed in an appropriate buffer at a temperature of 20xc2x0-70xc2x0 C., generally in a volume of 50 xcexcl or less with 0.1-10 xcexcg DNA.
The recombinant host cell is prepared, in accordance with the invention, by transforming a host cell with a replicable expression vector of the invention under transforming conditions. Suitable transforming conditions are conventional and are described in, for example, Sambrook et al., cited above, or xe2x80x9cDNA Cloningxe2x80x9d Vol. II, D. M. Glover ed., IRL Press Ltd, 1985.
The choice of transforming conditions is determined by the host cell. Thus, a bacterial host such as E. coli, may be treated with a solution of CaCl2 (Cohen et al., Proc. Nat. Acad. Sci., 1973, 69, 2110) or with a solution comprising a mixture of RbCl, MnCl2, potassium acetate and glycerol, and then with 3-[N-morpholino]-propane-sulphonic acid, RbCl and glycerol or by electroporation as for example described by Bio-Rad Laboratories, Richmond, Calif., USA, manufacturers of an electroporator. Mammalian cells in culture may be transformed by calcium co-precipitation of the vector DNA onto the cells or by using cationic liposomes.
The invention also extends to a host cell transformed with a replicable expression vector of the invention.
Culturing the transformed host cell under conditions permitting expression of the DNA polymer is carried out conventionally, as described in, for example, Sambrook et al., and xe2x80x9cDNA Cloningxe2x80x9d cited above. Thus, preferably the cell is supplied with nutrient and cultured at a temperature below 45xc2x0 C.
The protein product is recovered by conventional methods according to the host cell. Thus, where the host cell is bacterial such as E. coli and the protein is expressed intracellularly, it may be lysed physically, chemically or enzymatically and the protein product isolated from the resulting lysate. Where the host cell is mammalian the product is usually isolated from the nutrient medium.
Where the host cell is bacterial, such as E. coli, the product obtained from the culture may require folding for optimum functional activity. This is most likely if the protein is expressed as inclusion bodies. There are a number of aspects of the isolation and folding process that are regarded as important. In particular, the polypeptide is preferably partially purified before folding, in order to minimise formation of aggregates with contaminating proteins and minimise misfolding of the polypeptide. Thus, the removal of contaminating E. coli proteins by specifically isolating the inclusion bodies and the subsequent additional purification prior to folding are important aspects of the procedure.
The folding process is carried out in such a way as to minimise aggregation of intermediate-folded states of the polypeptide. Thus, careful consideration needs to be given to, among others, the salt type and concentration, temperature, protein concentration, redox buffer concentrations and duration of folding. The exact condition for any given polypeptide generally cannot be predicted and must be determined by experiment.
There are numerous methods available for the folding of proteins from inclusion bodies and these are known to the skilled worker in this field. The methods generally involve breaking all the disulphide bonds in the inclusion body, for example with 50 mM 2-mercaptoethanol, in the presence of a high concentration of denaturant such as 8M urea or 6M guanidine hydrochloride. The next step is to remove these agents to allow folding of the proteins to occur. Formation of the disulphide bridges requires an oxidising environment and this may be provided in a number of ways, for example by air, or by incorporating a suitable redox system, for example a mixture of reduced and oxidised glutathione.
Preferably, the inclusion body is solubilised using 8M urea, in the presence of mercaptoethanol, and protein is folded, after initial removal of contaminating proteins, by addition of cold buffer. A preferred buffer is 20 mM ethanolamine containing 1 mM reduced glutathione and 0.5 mM oxidised glutathione. The folding is preferably carried out at a temperature in the range 1 to 5xc2x0 C. over a period of 1 to 4 days.
If any precipitation or aggregation is observed, the aggregated protein can be removed in a number of ways, for example by centrifugation or by treatment with precipitants such as ammonium sulphate. Where either of these procedures are adopted, monomeric polypeptide is the major soluble product.
If the bacterial cell secretes the protein, folding is not usually necessary.
Peptide linkages in the derivatives of the invention may be made chemically or biosynthetically by expression of appropriate coding DNA sequences. Non peptide linkages may be made chemically or enzymatically by post-translational modification.
The polypeptide portion of the derivatives of the invention may be prepared by expression in suitable hosts of modified genes encoding the soluble polypeptide of the invention plus one or more peptide membrane binding elements and optional residues such as cysteine to introduce linking groups to facilitate post translational derivatisation with additional membrane binding elements.
The polypeptide portion of the derivative of the inversion may include a C-terminal cysteine to facilitate post translational modification. Expression in a bacterial system is preferred for proteins of moderate size (up to xcx9c70 kDa) and with  less than xcx9c8 disulphide bridges. More complex proteins for which a free terminal Cys could cause refolding or stability problems may require stable expression in mammalian cell lines (especially CHO). This will also be needed if a carbohydrate membrane binding element is to be introduced post-translationally. The use of insect cells infected with recombinant baculovirus encoding the polypeptide portion is also a useful general method for preparing more complex proteins and will be preferred when it is desired to carry out certain post-translational processes (such as palmitoylation) biosynthetically (see for example, M. J. Page et al J.Biol.Chem. 264, 19147-19154, 1989).
A preferred method of handling proteins C-terminally derivatised with cysteine is as a mixed disulphide with mercaptoethanol or glutathione or as the 2-nitro, 5-carboxyphenyl thio-derivative as generally described below in Methods.
Peptide membrane binding elements may be prepared using standard solid state synthesis such as the Merrifield method and this method can be adapted to incorporate required non-peptide membrane binding elements such as N-acyl groups derived from myristic or palmitic acids at the N terminus of the peptide. In addition activation of an amino acid residue for subsequent linkage to a protein can be achieved during chemical synthesis of such membrane binding elements. Examples of such activations include formation of the mixed 2-pyridyl disulphide with a cysteine thiol or incorporation of an N-haloacetyl group. Peptides can optionally be prepared as the C-terminal amide.
The derivatives of the invention may utilise a peptide membrane binding element comprising one or more derivatisations selected from:
a terminal cysteine residue optionally activated at the thiol group;
an N-haloacetyl group (where halo signifies chlorine, bromine or iodine) located at the N-terminus of the the peptide or at an xcex5-amino group of a lysine residue;
an amide group at the C-terminus; and
a fatty acid N-acyl group at the N-terminus or at an xcex5-amino group of a lysine residue.
Chemical bridging groups include those described in EP0109653 and EP0152736. The bridging group is generally of the formula:
-A-R-B-xe2x80x83xe2x80x83(V)
in which each of A and B, which may be the same or different, represents xe2x80x94COxe2x80x94, xe2x80x94C(xe2x95x90NH2+)xe2x80x94, maleimido, xe2x80x94Sxe2x80x94 or a bond and R is a bond or a linking group containing one or more xe2x80x94(CH2)xe2x80x94 or meta- or para-disubstituted phenyl units.
Were the polypeptide portion of the derivative of the invention and a peptide membrane binding element both include a C-terminal cysteine the chemical bridging group will take the form xe2x80x94Sxe2x80x94Sxe2x80x94. The bridge is generated by conventional disulphide exchange chemistry, by activating a thiol on one polypeptide and reacting the activated thiol with a free thiol on the other polypeptide. Such activation procedures make use of disulphides which form stable thiolate anions upon cleavage of the Sxe2x80x94S linkage and include reagents such as 2,2xe2x80x2 dithiopyridine and 5,5xe2x80x2-dithio(2-nitrobenzoic acid, DTNB) which form intermediate mixed disulphides capable of further reaction with thiols to give stable disulphide linkages.
R may include moieties which interact with water to maintain the water solubility of the linkage and suitable moieties include xe2x80x94COxe2x80x94NHxe2x80x94, xe2x80x94COxe2x80x94NMexe2x80x94, xe2x80x94Sxe2x80x94Sxe2x80x94, xe2x80x94CH(OH)xe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94CO2xe2x80x94, xe2x80x94(CH2CH2xe2x80x94O)mxe2x80x94 and xe2x80x94CH(COOH)xe2x80x94 where m is an integer of 2 or more.
Examples of R include xe2x80x94(CH2)rxe2x80x94, xe2x80x94(CH2)pxe2x80x94Sxe2x80x94Sxe2x80x94(CH2)qxe2x80x94 and xe2x80x94(CH2)pxe2x80x94CH(OH)xe2x80x94CH(OH)xe2x80x94(CH2)qxe2x80x94, in which r is an integer of at least 2, preferably at least 4 and p and a are independently integers of at least 2.
The bridging group of formula (V) may be derived from a linking agent of formula (VI):
X-R1-Yxe2x80x83xe2x80x83(VI)
in which R1 is a linking group containing one or more xe2x80x94(CH2)xe2x80x94 units and X and Y are functional groups reactable with surface amino acid groups, preferably a lysine or cysteine group, or the N-terminal amino group, or a protein attachment group.
Preferred agents are those where X and Y are different, known as heterobifunctional agents. Each end of the agent molecule is reacted in turn with each polypeptide to be linked in separate reactions. Examples of heterobifunctional agents of formula (VI) include:
N-succinimidyl 3-(2-pyridyldithio)propionate
succinimidyl 4-(N-maleimido)caproate
3-(2-pyridyl)methyl propionimidate hydrochloride
In each case Y is capable of reacting with a thiol group on a polypeptide, which may be a native thiol or one introduced as a protein attachment group.
The protein attachment group is a functionality derived by modification of a polypeptide with a reagent specific for one or more amino acid side chains, and which contains a group capable of reacting with a cleavable section on the other polypeptide. An example of a protein attachment group is a thiol group. An example of a cleavable section is a disulphide bond. Alternatively the cleavable section may comprise an xcex1, xcex2 dihydroxy function.
As an example, the introduction of a free thiol function by reaction of a polypeptide with 2-iminothiolane, N-succinimidyl 3-(2-pyridyldithio)propionate (with subsequent reduction) or N-acetyl homocysteine thiolactone will permit coupling of the protein attachment group with a thiol-reactive B structure. Alternatively, the protein attachment group can contain a thiol-reactive entity such as the 6-maleimidohexyl group or a 2-pyridyl-dithio group which can react with a free thiol in X. Preferably, the protein attachment group is derived from protein modifying agents such as 2-iminothiolane that react with lysine xcex5-amino groups in proteins.
When X represents a group capable of reacting directly with the amino acid side chain of a protein, it is preferably an N-succinimidyl group. When X represents a group capable of reacting with a protein attachment group, it is preferably a pyridylthio group.
In the above processes, modification of a polypeptide to introduce a protein attachment group is preferably carried out in aqueous buffered media at a pH between 3.0 and 9.0 depending on the reagent used. For a preferred reagent, 2-iminothiolane, the pH is preferably 6.5-8.5. The concentration of polypeptide is preferably high ( greater than 10 mg/ml) and the modifying reagent is used in a moderate (1.1- to 5-fold) molar excess, depending on the reactivity of the reagent. The temperature and duration of reaction are preferably in the range 0xc2x0-40xc2x0 C. and 10 minutes to 7 days. The extent of modification of the polypeptide may be determined by assaying for attachment groups introduced.
Such assays may be standard protein chemical techniques such as titration with 5,5xe2x80x2-dithiobis-(2-nitrobenzoic acid). Preferably, 0.5-3.0 moles of protein attachment group will be introduced on average per mole of polypeptide. The modified polypeptide may be separated from excess modifying agents by standard techniques such as dialysis, ultrafiltration, gel filtration and solvent or salt precipitation. The intermediate material may be stored in frozen solution or lyophilised.
Where a protein attachment group is introduced in this way, the bridging group (V) will be formed from a reaction of the linking agent (VI) and the protein attachment group.
The polypeptides to be linked are reacted separately with the linking agent or the reagent for introducing a protein attachment group by typically adding an excess of the reagent to the polypeptide, usually in a neutral or moderately alkaline buffer, and after reaction removing low molecular weight materials by gel filtration or dialysis. The precise conditions of pH, temperature, buffer and reaction time will depend on the nature of the reagent used and the polypeptide to be modified. The polypeptide linkage reaction is preferably carried out by mixing the modified polypeptides in neutral buffer in an equimolar ratio. Other reaction conditions e.g. time and temperature, should be chosen to obtain the desired degree of linkage. If thiol exchange reactions are involved, the reaction should preferably be carried out under an atmosphere of nitrogen. Preferably, UV-active products are produced (eg from the release of pyridine 2-thione from 2-pyridyl dithio derivatives) so that coupling can be monitored.
After the linkage reaction, the polypeptide conjugate can be isolated by a number of chromatographic procedures such as gel filtration, ion-exchange chromatography, affinity chromatography or hydrophobic interaction chromatography. These procedures my be either low pressure or high performance variants.
The conjugate may be characterised by a number of techniques including low pressure or high performance gel filtration, SDS polyacrylamide gel electrophoresis or isoelectric focussing.
Membrane binding elements which are fatty acid derivatives are attached post translationally to a peptidic membrane binding element, preferably at the terminus of the polypetide chain. Preferably, where the recombinant polypeptide portion of the derivative of the invention contains the peptide membrane binding element, it has a unique cysteine for coupling to the fatty acid derivative. Where the recombinant polypeptide has a cysteine residue, a thiol-derivative of the fatty acid is added to the refolded recombinant protein at a late stage in purification (but not necessarily the final stage) and at a reagent concentration preferably below the critical micelle concentration. One of the fatty acid derivative and the recombinant peptide will have the thiol group activated as described above for thiol interchange reactions. The fatty acid derivative is preferably a fatty acyl derivative of an aminoC2-6alkane thiol (optionally C-substituted) such as N-(2-myristoyl)aminoethanethiol or N-myristoyl L-cysteine.
Suitable examples of hydrophilic synthetic polymers include polyethyleneglycol (PEG), preferably xcex1,xcfx89 functionalised derivatives, more preferably xcex1-amino, xcfx89-carboxy-PEG of molecular weight between 400 and 5000 daltons which are linked to the polypeptide for example by solid-phase synthesis methods (amino group derivatisation) or by thiol-interchange chemistry.
Membrane binding elements derived from ligands of known integral membrane proteins, either amino acid sequences or carbohydrates, may be generated by post-translational modification using the glycosylation pathways of eukaryotic cells targeted to N-linked glycosylation sites in the peptide sequence.
Convenient generic final stage purification strategies are hydrophobic interaction chromatography (HIC) on C2-C8 media and cation exchange chromatography for separation of derivatised and underivatised proteins into which a hydrophobic electrostatic switch combination has been inserted.
In a further aspect, therefore, the invention provides a process for preparing a derivative according to the invention which process comprises expressing DNA encoding the polypeptide portion of said derivative in a recombinant host cell and recovering the product and thereafter post translationally modifying the polypeptide to chemically introduce membrane binding elements.
The invention also extends to DNA encoding the polypeptide portion of the derivative and to replicable expression vectors and recombinant host cells containing the DNA.
The polypeptide or derivative of this invention is useful in the treatment or diagnosis of many complement-mediated or complement-related diseases and disorders including, but not limited to, those listed below.
Disease and Disorders Involving Complement
Neurological Disorders
multiple sclerosis
stroke
Guillain Barre Syndrome
traumatic brain injury
Parkinson""s disease
allergic encephalitis
Alzheimer""s disease
Disorders of Inappropriate or Undesirable Complement Activation
haemodialysis complications
hyperacute allograft rejection
xenograft rejection
corneal graft rejection
interleukin-2 induced toxicity during IL-2 therapy
paroxysmal nocturnal haemoglobinuria
Inflammatory Disorders
inflammation of autoimmune diseases
Crohn""s Disease
adult respiratory distress syndrome
thermal injury including burns or frostbite
uveitis
psoriasis
asthma
acute pancreatitis
Kawasaki""s disease
Post-Ischemic Reperfusion Conditions
myocardial infarction
balloon angioplasty
atherosclerosis (cholesterol-induced) and restenosis
hypertension
post-pump syndrome in cardiopulmonary bypass or renal haemodialysis
renal ischemia
intestinal ischaemia
Infectious Diseases or Sepsis
multiple organ failure
septic shock
Immune Complex Disorders and Autoimmune Diseases
rheumatoid arthritis
systemic lupus erythematosus (SLE)
SLE nephritis
proliferative nephritis
glomerulonephritis
haemolytic anemia
myasthenia gravis
Reproductive Disorders
antibody- or complement-mediated infertility
Wound Healing
The present invention is also directed to a pharmaceutical composition comprising a therapeutically effective amount of a polypeptide or derivative, as above, and a pharmaceutically acceptable carrier or excipient.
The present invention also provides a method of treating a disease or disorder associated with inflammation or inappropriate complement activation comprising administering to a subject in need of such treatment a therapeutically effective amount of a polypeptide or derivative of this invention.
In the above methods, the subject is preferably a human.
An effective amount of the polypeptide or derivative for the treatment of a disease or disorder is in the dose range of 0.01-100 mg/kg; preferably 0.1 mg-10 mg/kg.
For administration, the polypeptide or derivative should be formulated into an appropriate pharmaceutical or therapeutic composition. Such a composition typically contains a therapeutically active amount of the polypeptide or derivative and a pharmaceutically acceptable excipient or carrier such as saline, buffered saline, dextrose, or water. Compositions may also comprise specific stabilising agents such as sugars, including mannose and mannitol, and local anaesthetics for injectable compositions, including, for example, lidocaine.
Further provided is the use of a polypeptide or derivative of this invention in the manufacture of a medicament for the treatment of a disease or disorder associated with inflammation or inappropriate complement activation.
In order to inhibit complement activation and, at the same time, provide thrombolytic therapy, the present invention provides compositions which further comprise a therapeutically active amount of a thrombolytic agent. An effective amount of a thrombolytic agent is in the dose range of 0.01-10 mg/kg; preferably 0.1-5 mg/kg. Preferred thrombolytic agents include, but are not limited to, streptokinase, human tissue type plasminogen activator and urokinase molecules and derivatives, fragments or conjugates thereof. The thrombolytic agents may comprise one or more chains that may be fused or reversibly linked to other agents to form hybrid molecules (EP-A-0297882 and EP 155387), such as, for example, urokinase linked to plasmin EP-A-0152736), a fibrinolytic enzyme linked to a water-soluble polymer (EP-A-0183503). The thrombolytic agents may also comprise muteins of plasminogen activators (EP-A-0207589). In a preferred embodiment, the thrombolytic agent may comprise a reversibly blocked in vitro fibrinolytic enzyme as described in U.S. Pat. No. 4,285,932. A most preferred enzyme is a p-anisoyl plasminogen-streptokinase activator complex as described in U.S. Pat. No. 4,808,405, and marketed under the Trademark EMINASE (generic name anistreplase, also referred to as APSAC; Monk et al., 1987, Drugs 34:25-49).
Routes of administration for the individual or combined therapeutic compositions of the present invention include standard routes, such as, for example, intravenous infusion or bolus injection. Active complement inhibitors and thrombolytic agents may be administered together or sequentially, in any order.
The present invention also provides a method for treating a thrombotic condition, in particular acute myocardial infarction, in a human or non-human animal. This method comprises administering to a human or animal in need of this treatment an effective amount of a polypeptide or derivative according to this invention and an effective amount of a thrombolytic agent.
Also provided is the use of a polypeptide or derivative of this invention and a thrombolytic agent in the manufacture of a medicament for the treatment of a thrombotic condition in a human or animal. Such methods and uses may be carried out as described in WO 91/05047.
This invention further provides a method for treating adult respiratory distress syndrome (ARDS) in a human or non-human animal. This method comprises administering to the patient an effective amount of a polypeptide or derivative according to this invention.
The invention also provides a method of delaying hyperacute allograft or hyperacute xenograft rejection in a human or non-human animal which receives a transplant by administering an effective amount of a polypeptide or derivative according to this invention. Such administration may be to the patient or by application to the transplant prior to implantation.
The invention yet further provides a method of treating wounds in a human or non-human animal by administering by either topical or parenteral e.g. intravenous routes, an effective amount of a polypeptide or derivative according to this invention.